["The Journal of Physiology, Volume 604, Issue 13, Page 5440-5457, 1 July 2026. ", "\nAbstract figure legend Graphical representation of methods. We implemented three biventricular geometric models (Zenger et al., 2020) with rule‐based myocardial fibre orientations (Bayer et al., 2018). We evaluated variability in the fibre orientation via four sets of parameter distributions to determine the role of the primary and imbrication angles (Streeter et al., 1969; Lombaert et al., 2012). We applied polynomial chaos expansion using UncertainSCI (Narayan et al., 2023; Tate et al., 2023) to model parameter variability and its impact on epicardial activation times simulated in CARPentry using the eikonal model (Augustin et al., 2016). Statistical moments, such as mean, standard deviation (STD) and global sensitivities, were calculated for epicardial activation times.\n\n\n\n\n\n\n\n\n\nAbstract\nPredictive models and computational simulations of cardiac electrophysiology depend on precise anatomical representations, including the local myocardial fibre structure. However, obtaining patient‐specific fibre information is challenging. In addition, the influence of physiological variability in fibre orientation on cardiac activation simulations is poorly understood. We implemented rule‐based algorithms to generate fibres and robust uncertainty quantification methods to determine model output variability with respect to ventricular activation sequences. We used polynomial chaos, which reduces computational demands by using an emulator to approximate the underlying forward model. Our study examined activation sequences in response to nine stimuli and five metrics quantifying essential features of the activation sequence. The results indicated that the primary fibre orientation impacts the overall spread of activation, which could impact more complex patterns of activation; however, there is minimal impact on the location of discrete activation features, such as breakthrough sites. For free wall stimuli, the standard deviation (STD) was highest near the stimulus site, diminishing with distance. Apical stimuli showed complementary STD patterns, with epicardial pacing maximizing STD in the right basal area and endocardial pacing in the left. Ventricular junction stimuli exhibited symmetrical STD patterns, low near the stimulus but increasing sharply towards the apex, peaking on the left in the apical region. Furthermore, variability in the imbrication or helix angle did not impact the activation sequences. We conclude that in many relevant modelling contexts, the variability in myocardial fibre orientation can play an important role in the resulting activation sequences and should be accounted for.\n\n\n\n\n\n\n\n\n\nKey points\nThe primary fibre orientation has modest impact on activation duration and location of most discrete activation features for ectopic stimuli, but introduces variability in activation sequence.\nFor free wall stimuli, the standard deviation (STD) was highest on the stimulated surface, indicating that deviations are largest in early activation and diminish as activation reaches remote heart regions.\nFor apical stimuli, activation patterns were insensitive to fibre orientation variations, but the STD had complementary maxima, strongly dependent on pacing surface. Epicardial pacing produced largest STD in right basal area while endocardial pacing affected left basal area, indicating strong dependence on fibre structure.\nFor stimuli at both anterior and posterior ventricular junctions, STD patterns were symmetrical, with low values near stimulus and sharp increases towards apex, peaking in left apical region.\nThe helical fibre orientation showed no relevant fluctuations in activation sequence.\n\n\n"]